Introduction

Hepatitis A is a non-enveloped RNA virus 27 to 32 nm in diameter. It has an icosahedral symmetry and belongs to the genus Hepatovirus of the Picornaviridae family. Infections with HAV can produce effects that range in severity from asymptomatic to death from fulminant hepatitis. Infections with HAV are typically self-limiting and do not result in chronic liver disease, however, outbreaks of HAV require considerable amounts of public health resources due to long incubation and shedding period after infection The virus in shed in the feces and peak fecal excretion, hence infectivity, occurs prior to the onset of symptoms (Lednar et al., 1985). These viruses are considered to be highly infectious and illness can be caused by as few as 10 viral particles (Teunis, 2008). Illnesses caused by transmission of these viruses in food have been epidemiologically-linked to three distinct classes: (i) cases associated with the consumption of ready-to-eat (RTE) foods contaminated by food workers; (ii) cases associated with the environmental contamination of produce, and (iii) cases associated with the consumption of molluscan shellfish harvested from contaminated water.

Significant concern of food-borne viruses on food safety and public health is the current limitations on matrix-associated virus detection. Detection in implicated foods is considered difficult because of the low level of viral contamination, inefficient extraction from food matrices, and the inability to efficiently enrich viruses-an aspect beneficial to most other bacterial detection methodologies. These factors necessitate that isolated virus particles be sufficiently concentrated in order to detect their presence in foods. Such limitations have hindered the FDA’s efforts to provide effective regulatory oversight in surveillance and outbreak investigations. Advances in molecular detection techniques e.g. reverse transcription (RT)-PCR and real-time RT-qPCR (Arnal, Ferre-Aubineau et al. 1999; Schwab, Neill et al. 2000; Sair, D'Souza et al. 2002) have been shown to offer specificity and sensitivity for food-borne pathogen detection. These methods are ideal for the detection of viruses with low titers. The use of these methods during outbreaks and routine surveillance would allow for faster sample throughput by quickly screening samples and allowing for more time to analyze "Cannot Rule Out" (CRO’s). In addition, at the time of this study, there were no multi-lab validated rapid protocols available for detection of enteric viruses in food.

Detection Method

The detection assay includes oligonucleotide primers and dual-labeled hydrolysis (Taqman-style) probes for the in vitro quantifiable detection of HAV. The HAV primers are from the 5’ untranslated region (UTR) of the genome and designed for detection of all genotypes of HAV (Gardner et al. 2003). This assay also incorporates an RNA internal amplification control (IAC) to monitor any potential matrix-derived inhibitory effects.

HAV Primers and Probe

All HAV probes and primers were commercially synthesized (Integrated DNA Technologies, Coralville, IA). The HAV probe is labeled 5’ with Cy5 reporter dye and 3’ with Iowa Black RQ as a quencher. The IAC probe was labeled 5’ with TxRed reporter dye and 3’ with Iowa Black RQ as a quencher. All primers and probes are hydrated in sterile primer TE buffer (see Appendix D) to 100µM concentration. Ten µM working stocks are prepared from the 100 µM stock solution and are stored at -20 °C in a frost free freezer.

Internal Control Primer, Probes and RNA

Table 1. Primer and Probe Sequences for HAV and Internal Amplification Control RNA

* Amount varies with concentration of IAC RNA. The amount of IAC template needs to be adjusted based on the prepared stock concentration to report Cycle threshold at about 20-25 PCR cycles when no inhibition is present in the reaction. The required concentration was provided to each participating laboratory.˜ With the addition of 1.5 mM MgCl2, the final concentration per reaction is 4.0mM MgCl2

Table 3. HAV RT-qPCR Cycling Conditions: Cepheid Smart Cycler II

Stage 1

Stage 2

Stage 3

Hold

Hold

3 Temperature Cycle

Temp

Sec

Optics

Temp

Sec

Optics

Repeat 45 times

50

3000

Off

95

900

Off

Temp

Sec

Optics

95

10

Off

53

25

Off

64

70

On

Qualitative data analysis.

On the SmartCycler II Instrument, set the following Analysis Settings for TxRd and Cy5 channels. Update analysis settings if they are changed before recording results.

Usage: Assay

Curve Analysis: Primary

Threshold Setting: Manual

Manual Threshold Fluorescence Units: 10.0

Auto Min Cycle: 5

Auto Max Cycle: 10

Valid Min Cycle: 3

Valid Max. Cycle: 60

Background subtraction: ON

Boxcar Avg. Cycles: 0

Background Min. Cycle: 5

Background Max. Cycle: 40

Max Cycles: 50

Primary fluorescence curves that cross the threshold will be recorded as "POS" and the cycle number when it crossed the threshold will be displayed in the Results Table view (Figure 1A). Negative results are shown as "NEG". The TxRd and Cy5 channels correlate to IAC and the HAV targets, respectively. Results can also be viewed graphically. For example, Figure 1B is a graphical view for the two channels for the Strain of HAV HM175/18f.

Negative samples:Sample is "negative" if the RT-qPCR negative control is negative for HAV, the RT-qPCR positive control is positive for HAV, the un-spiked sample is negative for HAV, and the internal amplification control (IAC) is positive.

No further analysis is needed.

Positive Samples:Sample is "positive" if the RT-qPCR negative control is negative for HAV, RT-qPCR positive control is positive for HAV, the spiked HAV RNA RT-PCR sample is positive for HAV, and the internal amplification control (IAC) is positive.

Note: If the negative RT-qPCR control sample demonstrates positive results crossing the Cy5 threshold or if the IAC is negative, the assay must be repeated.

Concentration and Extraction Methods

The concentration and extraction protocol uses ultracentrifugation to concentrate HAV eluted from green onion (Figure 2) . Concentration and extraction of viruses from produce can be difficult due to the increased presence of polysaccharides. The use of the QIAshredder and a slight modification of the QIAamp viral RNA extraction kit produce a RNA viral concentrate with minimal inhibition. Murine norovirus (MNV), ATCC PTA-5935, is used as an extraction control to access the overall performance of the method.

Add 25 µl of heated Buffer AVE to column. Pipette the eluted 35 µl back to the top of the column. Close the tube gently, and centrifuge for 1 min at ≥8000 × g (≥10,000 rpm)

Label samples, use immediately, place elute on ice, or freeze at -70 °C for long term storage

RT-qPCR for Murine Norovirus (MNV) extraction control

This method will assess the recovery of murine norovirus from spiked samples and determine if the extraction was performed correctly. The test utilizes IAC primers and probe that are multiplexed (simultaneously amplified) with MNV primers and probe for each RNA sample. The IAC adds value to the assay by confirming that amplifiable RNA is present in samples that test PCR-negative for MNV. The IAC also verifies efficient nucleic acid extraction and removal of RT-PCR inhibitors.

* Amount varies with concentration of IAC RNA. The amount of IAC template needs to be adjusted based on the prepared stock concentration to report Cycle threshold at about 20-25 PCR cycles when no inhibition is present in the reaction.+ Final concentration for 25 µl reaction is 4.0mM.

Table 6. MNV RT-qPCR Cycling Conditions: Cepheid Smart Cycler II

Stage 1

Stage 2

Stage 3

Hold

Hold

3 Temperature Cycle

Temp

Sec

Optics

Temp

Sec

Optics

Repeat 45 times

50

3000

Off

95

900

Off

Temp

Sec

Optics

95

15

Off

55

20

Off

62

60

On

Qualitative data analysis

On the SmartCycler II Instrument, set the following Analysis Settings for TxRed and Cy5 channels. Update analysis settings if they are changed before recording results.

Usage: Assay

Curve Analysis: Primary

Threshold Setting: Manual

Manual Threshold Fluorescence Units: 10.0

Auto Min Cycle: 5

Auto Max Cycle: 10

Valid Min Cycle: 3

Valid Max. Cycle: 60

Background subtraction: ON

Boxcar Avg. Cycles: 0

Background Min. Cycle: 5

Background Max. Cycle: 40

Max Cycles 50

Primary fluorescence curves that cross the threshold will be recorded as "POS" and the cycle number when it crossed the threshold will be displayed in the Results Table view (Figure 3). Negative results are shown as "NEG". The TxRd and Cy5 channels correlate to IAC and the MNV targets, respectively. Results can also be viewed graphically. For example, Figure 3 is a graphical view for the two channels for the MNV and IAC.

Figure 3

Interpretation of real-time RT-PCR Results (Smart Cycler II)

Positive Samples:Extraction controls are considered positive when value crosses threshold for Cy5 fluorescence channel for MNV spiked samples, IAC is positive within acceptable ranges, and positive MNV RT-qPCR control is positive and the negative RT-qPCR control is negative.

Negative samples:Sample is "negative" when the MNV spiked control sample shows no detection for Cy5 fluorescence channel, the negative RT-qPCR control is negative, the positive MNV RT-qPCR control is positive for MNV, and the internal amplification control (IAC) is positive.

Invalid Samples:

If the negative RT-qPCR control sample demonstrates positive results crossing the Cy5 threshold or if the IAC is negative, the RT-PCR assay must be repeated.

The average of the Internal Amplification Control Ct values for the sample is more than 4 Ct s greater than the Negative Control Internal Amplification Control Ct value; the RT-PCR assay must be repeated using RNA from a newly extracted saved tube. If the repeat of the newly extracted sample yields average IAC Ct values greater than 4, the sample must be repeated from the beginning using additional food sample.

Detection of HAV in green onion.

The HAV multiplex assay is used to detect HAV in green onion RNA extracts. In figure 4, the graph demonstrates the TxRd and Cy5 channels correlating to the HAV and the IAC targets, respectively. Probable positive HAV samples or Cannot Rule Out (CRO) occurs when the primary fluorescence curve crosses the threshold for HAV and the IAC is positive. Sample data is considered invalid if: (1) the average of the HAV internal control Ct for the samples is ≥3.5 Ct values compared to the negative control and the MNV extraction control is not detected, and (2) false positive or false negative controls.

Figure 4.

Interpretation of real-time RT-PCR Results (Smart Cycler II)

Interpretation of Results:For this HAV assay Cy5 is the HAV probe fluorescent label and that Texas Red (TxR) is the internal amplification control (IAC) probe fluorescent label.

Negative samples:Sample is "negative" if the RT-qPCR negative control is negative for HAV, the RT-qPCR positive control is positive for HAV, the un-spiked sample (if included) is negative for HAV, the unknown is negative for HAV, and the internal amplification control (IAC) is positive. No further analysis is needed.

Positive Samples:Sample is "positive" if the RT-qPCR negative control is negative for HAV, RT-qPCR positive control is positive for HAV, the unknown or spiked HAV RNA RT-PCR sample is positive for HAV, and the internal amplification control (IAC) is positive.

Invalid Samples:

If the negative RT-qPCR control sample demonstrates positive results crossing the Cy5 threshold or if the IAC is negative, the RT-PCR assay must be repeated.

The average of the Internal Amplification Control Ct values for the sample is more than 4 Ct’s greater than the Negative Control Internal Amplification Control Ct value, the RT-PCR assay must be repeated using RNA from a newly extracted saved tube. If the repeat of the newly extracted sample yields average IAC Ct values greater than 4, the sample must be repeated from the beginning using additional food sample.

Probable positive HAV samples or Cannot Rule Out (CRO):If the HAV sample is positive and the internal controls, negative controls, positive controls, and extraction controls are correct. A CRO protocol is available for use when required.